Pharmaceutical compositions of chitosan with type-A gelatin

ABSTRACT

A drug delivery composition for nasal administration is provided which comprises the antiviral agent ICAM-1 and a bioadhesive material. The bioadhesive material may be a chitosan solution, a liquid formulation comprising a polymeric material or a plurality of bioadhesive microspheres. The polymeric material is preferably gellan gum or alginate. The microspheres may comprise starch, chitosan, hyaluronic acid, or gelatin.

Priority is claimed under 35 U.S.C. §119 to PCT/GB98/00108, filed Jan.14, 1998, which corresponds to GB 97/00624.1 filed Jan. 14, 1997.

This invention relates to novel drug delivery compositions which providefor the improved uptake of therapeutic agents across mucosal surfaces.

Polar drugs, including high molecular weight peptides, proteins andpolysaccharides, are typically not effectively absorbed across mucosalmembranes, such as the gastrointestinal tract, the eye, the vagina, thenasal cavity or the rectum. Such molecules are thus normally only givenby injection, which inevitably gives rise to well known problemsassociated with patient compliance, the cost of treatment, as well asthe potentially harmful effects, such as phlebitis and pain, of theinjection.

It is well known in the literature that the absorption of polarmolecules across mucosal membranes may be greatly improved if they areadministered in combination with so-called “absorption enhancers”.Examples of absorption enhancers which have been described in theliterature include non-ionic surfactants, cyclodextrins, pholspholipidsand bile salts. (For a review see Davis et al (eds.), Delivery Systemsfor Peptide Drugs, Plenum Press, New York, 1987; and Lee (ed.), Peptideand Protein Delivery, Marcel Dekker Inc., New York, 1991.)

EP-A-023 359 and EP-A-122 023 describe powdery pharmaceuticalcompositions for application to the nasal mucosa, as well as methods forthe administration of such compositions. The pharmaceutical compositionsallow polypeptides and derivatives thereof to be effectively absorbedthrough the nasal mucosa. Similarly, U.S. Pat. No. 4,226,849 describes amethod for administering a powdery medicament to the nasal mucosa, inwhich the preferred composition has mucoadhesive properties.

Formulations based on microspheres for mucosal delivery have beendescribed in WO 88/09163. The formulations contain certain enhancers toaid effective penetration of the mucosa by the drug. WO 89/03207describes microsphere formulations which do not require an enhancer.

Chitosan is a derivative of chitin or poly-N-acetyl-D-glucosamine inwhich the greater proportion of the N-acetyl groups have been removedthrough hydrolysis. It is available from several suppliers includingPronova, Drammen, Norway, and, depending on the grade selected, issoluble in water and/or aqueous acid up to pH values of between 6.0 and7.0.

Chitosan has previously been used to precipitate proteinaceous materialand to make surgical sutures. It has also been employed previously inoral drug formulations in order to improve the dissolution of poorlysoluble drugs (see Sawavanagi et al, Chem. Pharm. Bull., 31 (1983)2062-2068) or for the sustained release of drugs by a process of slowerosion from a hydrated compressed matrix (Nagai et al, Proc. Jt. USJpn. Semin. Adv. Chitin Chitosan Relat. Enzymes, 21-39, Zikakis J. P.(ed.), Academic Press, Orlando, 1984).

WO 90/09780 describes a composition comprising a drug and a polycationicsubstance (e.g. chitosan) that promotes the transport of the drug acrossmucosal membranes. The composition may also comprise microspheres of thepolycationic substance.

WO 96/05810 describes a composition comprising a pharmacologicallyactive compound and particles, preferably powders or microspheres, ofchitosan or a chitosan derivative or salt, where the particles areeither solidified or partially cross-linked such that they have azeta-potential of between +0.5 and +50 mV. Solidified particles are madeby treating particles made from a water soluble chitosan salt with analkaline agent, such as sodium hydroxide, in non-acid containing waterto render them insoluble.

Chitosan microspheres have also been produced for use in enhancedchromatographic separation (Li Q. et al, Biomater. Artif. CellsImmobilization Biotechnology, 21 (1993) 391-398), for the topicaldelivery of drugs (Machida Y., Yakugaku Zasshl., 113 (1993) 356-368),for drug targeting after injection (Ohya Y et al, J. Microencap., 10(1993) 1-9), as an implantable controlled release delivery system(Jameela and Jayakrishnan, Biomaterials, 16 (1995) 769-775) and for thecontrolled release of drugs (see Bodmeier R. et al, Pharm. Res., 6(1989) 413-417 and Chithambara et al, J. Pharm. Pharmacol., 44 1992,283-286).

EP 454044 and EP 486959 describe polyelectrolyte microparticles orpolysaccharide microspheres, including chitosan microspheres, for use inthe controlled release of drugs. Chitosan microspheres crosslinked withglutaraldehyde have also been described in JP 539149.

Gelatin is a purified protein obtained either by partial acid hydrolysis(type A) or by partial alkaline hydrolysis (type B) of animal collagen.Type A gelatin is cationic with an isoelectric point between pH valuesof 7 and 9, whereas type B gelatin is anionic with an isoelectric pointbetween pH values of 4.7 and 5. Gelatin is known to swell and softenwhen immersed in cold water, eventually absorbing between 5 and 10 timesits own weight in water. It is soluble in hot water, forming a gel oncooling. Gelatin is used as a haemostatic in surgical procedures as anabsorbable film or sponge, which can absorb many times its own weight inblood. It is also employed as a plasma substitute, and may be used inthe preparation of pastes, pastilles, suppositories, tablets and hardand soft capsule shells for oral formulations.

The production of gelatin microspheres has been widely described in theliterature. Gelatin microspheres have been produced by an emulsificationmethod involving crosslinking with glutaraldehyde, producingmicrospheres of less than 2 μm in diameter (Tabata and Ikada, Pharm.Res. 6 (1989) 422-427). Cortesi et al (Int. J. Pharm. 105 (1994)181-186), Natruzzi et al (J. Microencapsulation, 11 (1994) 294-260) andEsposito et al (Int. J. Pharm., 117 (1995) 151-158) have reported theproduction of microspheres of a mean diameter of 22 μm using acoacervation emulsification method. Microspheres as produced by thelatter processes were not crosslinked. Microspheres of a smaller sizehave been produced according to a similar method by Esposito et al(Pharm. Sci. Commun. 4 (1994) 239-246). The type of gelatin (A or B)used in these studies was not specified.

The production of microspheres by complexation, between a negativelycharged material such as alginate and a positively charged chitosan hasbeen described in the literature. For example. Polk et al, J. Pharm.Sci., 83 (1994) 178-185) describes the production of clhitosan-alginatemicrospheres by the addition of an alginate solution to a solution ofchitosan and calcium ions. The highest concentration of chitosan used inthe microsphere formulations was 5.2% w/w. Similarly, the formation ofcomplex coacervates between oppositely charged polyions, namely apositively charged chitosan and a negatively charged type B gelatin hasbeen described by Remunan-Lopez and Bodmeier (Int. J. Pharm. 135 (1996)63-72). These workers found the optimum chitosan:gelatin ratio to be inthe range 1:10 to 1:20. The coacervate was obtained in a dry form bydecanting the supernatant after centrifugation and drying at 60° C. Wehave now found, surprisingly, that microparticles, produced from acombination of a chitosan and a cationic type A gelatin, possessparticularly advantageous properties, which enable the improvedtransport of therapeutic agents, including polar drugs, across mucosalsurfaces such as the nasal cavity.

Thus, according to a first aspect of the invention there is provided acomposition comprising a mixture of chitosan and type A. cationic,gelatin, together with a therapeutic agent (hereinafter referred to as“the compositions according to the invention”).

By “mixture of chitosan and type A gelatin” we include any compositioncomprising a chitosan, as defined hereinafter, and a type A gelatin, asdefined hereinafter, whether a physical and/or chemical associationbetween these two constituents exists or not.

The term “chitosan” will be understood by those skilled in the art toinclude all derivatives of chitin, or poly-N-aceryl-D-glucosamine(including all polyglucosamine and oligomers of glucosamine materials ofdifferent molecular weights), in which the greater proportion of theN-acetyl groups have been removed through hydrolysis. We prefer that thechitosan has a positive charge.

Chitosan, chitosan derivatives or salts (e.g. nitrate, phosphate,sulphate, hydrochloride, glutamate, lactate or acetate salts) ofchitosan may be used. We use the term chitosan derivatives to includeester, ether or other derivatives formed by bonding of acyl and/or alkylgroups with OH groups, but not the NH₂ groups, of chitosan. Examples areO-alkyl ethers of chitosan and 0-acyl esters of chitosan. Modifiedchitosans, particularly those conjugated to polyethylene glycol, areincluded in this definition. Low and medium viscosity chitosans (forexample CL113, G210 and CL110) may be obtained from various sources,including Pronova Biopolymer, Ltd., UK; Seigagaku America Inc.,Maryland, USA; Meron (India) Pvt, Ltd., India; Vanson Ltd, Virginia,USA; and AMS Biotechnology Ltd., UK. Suitable derivatives include thosewhich are disclosed in Roberts, Chitin Chemistry, MacMillan Press Ltd.,London (1992).

The chitosan or chitosan derivative or salt used preferably has amolecular weight of 4,000 Dalton or more, preferably in the range 25,000to 2,000,000 Dalton, and most preferably about 50,000 to 300,000 Dalton.Chitosans of different low molecular weights can be prepared byenzymatic degradation of chitosan using chitosanase or by the additionof nitrous acid. Both procedures are well known to those skilled in theart and are described in recent publications (Li et al. (1995) PlantPhysiol. Biochem. 33, 599-603; Allan and Peyron, (1995) CarbohydrateResearch 277, 257-272; Damard and Cartier, (1989) Int. J. Biol.Macromol. 11, 297-302).

Preferably, the chitosan is water-soluble and may be produced fromchitin by deacetylation to a degree of greater than 40%, preferablybetween 50% and 98%, and more preferably between 70% and 90%. Particulardeacetylated chitosans which may be mentioned include the “Sea Cure®”series of chitosan glutamates available from Protan Biopolymer A/S,Drammen, Norway.

The term “type A gelatin” includes all cationic proteins which are, ormay be, obtained by partial acid hydrolysis of animal collagen, andexcludes type B gelatins.

Although the compositions according to the invention may be prepared ina variety of physical forms using techniques which will be well known tothe skilled person, we prefer that the compositions are in the form ofmicroparticles. The term “microparticles” includes microspheres,microcapsules and powders. However, we prefer that the microparticlesare microspheres.

We have found, surprisingly, that when the compositions according to theinvention are provided in the form of microparticles, suchmicroparticles retain a positive charge and may provide for the improvedtransport of polar drugs across, or for the improved presentation ofvaccines to muscosal surfaces, such as the nasal cavity, to such anextent that the effect is superior to that obtained for a chitosansolution, or microparticles produced from chitosan or type A gelatinalone (e.g. soluble (spray dried) chitosan microsphercs and gelatinmicrospheres). The effect is also similar to that obtained for partiallyaldehyde crosslinked chitosan microspheres, yet the compositionsaccording to the invention are sufficiently hard/solid not to requirecrosslinking. We have further found that the flow properties of thesechitosan/type A gelatin microparticles are superior to those of spraydried chitosan microspheres and crosslinked chitosan microspheres.

The microparticles may be prepared by spray drying, emulsification,solvent evaporation, precipitation or other methods known to a personskilled in the art. The therapeutic agent can be incorporated into themicroparticles during their production or sorbed onto the microparticlesafter their production.

When the compositions according to the invention are in the form ofmicrospheres, they may be prepared using for example eitheremulsification or spray drying techniques.

When microsphercs are prepared by spray drying, a warm mixture ofchitosan and type A gelatin is spray dried with instant cooling of theresultant microspheres. The therapeutic agent may be incorporated byadsorbing onto the surface of the microspheres by freeze drying or spraydrying a suspension of the microspheres with the therapeutic agent, orby physically or mechanically mixing the dried microspheres with thetherapeutic agent.

However, we have found that microspheres may advantageously be preparedby warming a solution of a chitosan mixed with type A gelatin which isthen emulsified and gelated by cooling. We have found that, inparticular, microspheres prepared in accordance with this techniqueexhibit the advantageous properties referred to hereinbefore.

In the emulsification technique, the chitosan may be dissolved in waterand mixed with type A gelatin under heating to 40° C. causing thegelatin to melt. This mixture may be emulsified, at a temperature abovethe melting point of the gelatin, in an organic medium (e.g. a vegetableoil, such as sunflower oil, soya oil, cotton seed oil or coconut oil),in the presence of an emulsifier with a low hydrophilic-lipophilicbalance (HLB) value. Such emulsifiers, which are useful for stabilisingwater-in-oil emulsions, are known to those skilled in the art (e.g. Span80). The microspheres may then be solidified by decreasing thetemperature of the emulsion to below 10° C. with stirring. Themicrospheres may then be harvested using conventional techniques, forexample by adding a pharmaceutically acceptable organic solvent, e.g.chilled acetone or petroleum ether, to the emulsion, centrifugation,washing and drying. The therapeutic agent may be incorporated into themicrospheres by adding it to the chitosan/gelatin mixture beforeemulsification. Alternatively, the therapeutic agent may be adsorbedonto the surface of the microspheres by freeze drying or by spray dryinga suspension of the microsphercs with the therapeutic agent, or byphysically or mechanically mixing the dried microspheres with thetherapeutic agent.

Thus, according to a further aspect of the invention there is provided adrug delivery composition in a form suitable for administration to amucosa comprising a therapeutic agent and microparticles made from amixture of chitosan and type A gelatin and where the agent is eitherincorporated into the particles during production or is adsorbed to thesurface of the particles, or is present as an admixture.

Microcapsules and powders may be made by modifying the process asdefined herein in accordance with techniques which are well known tothose skilled in the art, or may be prepared in accordance with othertechniques which will be well known to those skilled in the art,including double emulsification processes.

According to a further aspect of the invention there is provided aprocess for the preparation of a composition according to the invention,which process comprises preparation of type A gelatin/chitosanmicroparticles (i.e. microparticles comprising a mixture of type Agelatin and chitosan) by a process of spray drying or by emulsification,which emulsification may comprise warming a solution of a chitosan mixedwith type A gelatin, emulsification and gelation by cooling.

The flow properties of the microparticles can be measured by methodsknown to those skilled in the art. One possible method involves themeasurement of the Hausner Ratio where a known weight of material ispoured into a measuring cylinder and the volume recorded. The cylinderis then tapped against a surface a specified number of times and thevolume again recorded. The poured and tapped densities are thendetermined and the Elausner Ratio=tapped density/poured densitycalculated. A ratio of <1.25 indicates a free flowing material while aratio of >1.5 indicates a poor flowing (cohesive) material. Anotherpossible method involves the measurement the Angle of Repose by pouringmaterial through a funnel held at a fixed height onto a piece of graphpaper until a cone is formed. The height (H) and the radius (R) of thecone is determined and the angle calculated (tan θ=H/R). An Angle ofRepose θ<300 indicates good flow properties while an Angle of Reposeθ>400 indicates very poor flow properties (James I. Wells,Pharmaceutical Preformulation, Ellis Horwood Series in PharmaceuticalTechnology, 1988).

The size of the microparticles, which includes microcapsules andespecially microspheres, is preferably in the range 1 to 200 μm, morepreferably 1 to 100 μm, as measured by e.g. light microscopy or sievefractionation.

The microparticles will consist of preferably between 50 and 95%, morepreferably between 70 and 90% and most preferably between 75 and 85% oftype A gelatin, and correspondingly between 50 and 5%, preferablybetween 30 and 10% and most preferably between 25 and 15% of chitosan,as measured in relation to the total amount of gelatin and chitosan inthe final composition (i.e. excluding therapeutic agent and otheringredients which may be included).

The term “therapeutic agent” includes drugs, genes (DNA) or geneconstructs, vaccines and components thereof (for example isolatedantigens or parts thereof) and monoclonal antibodies. For applicationsemploying such materials as genes, gene constructs, vaccines andmonoclonal antibodies, the microparticles can be used to enhance thedelivery of the therapeutic agent into the mucosal tissue for enhancedtherapeutic effect, for example presentation of an antigen to theunderlying lymphoid tissue, and/or transfection of the cells in themucosal lining.

Preferably the therapeutic agent is a polar drug. By “polar drugs” wemeans molecules with a partition coefficient (octanol—water system) ofless than 50.

The compositions may be used with therapeutic agents selected from thefollowing non-exclusive list: insulin, PTH (parathyroid hormone), PTHanalogues, PTHrP (human parathyroid hormone peptide), calcitonins (forexample porcine, human, salmon, chicken or eel) and syntheticmodifications thereof, enkephalins, LHRH (luteinising hormone releasinghormone) and analogues (nafarelin, buserelin, leuprolide, goserelin),glucagon, TRH (thyrotropine releasing hormone), vasopressin,desmopressin, growth hormone, heparins, GHRH (growth hormone releasinghormone), CCK (cholecystokinin), THF (thymic humoral factor), CGRP(calcitonin gene related peptide), atrial natriuretic peptide,nifedipine, metoclopramide, ergotamine, pizotizin, pentamidine andvaccines (particularly but not limited to AIDS vaccines, measlesvaccines, rhinovirus Type 13 and respiratory syncytial virus vaccines,influenza vaccines, pertussis vaccines, meningococcal vaccines, tetanusvaccines, diphtheria vaccines, cholera vaccines and DNA vaccines (e.g.one containing a plasmid DNA coding for a suitable antigen)).

Further therapeutic agents include but are not limited to: antibioticsand antimicrobial agents, such as tetracycline hydrochloride,leucomycin, penicillin, penicillin derivatives, erythromycin,sulphathiazole and nitrofurazone; anti-migraine compounds, such asnaratriptan, sumatriptan, alnitidan or other 5-HT1 agonists;vasoconstrictors, such as phenylephedrine hydrochloride,tetrahydrozoline hydrochloride, naphazolinie nitrate, oxymetazolinehydrochloride and tramazoline hydrochloride; cardiotonics, such asdigitalis and digoxin; vasodilators, such as nitroglycerine andpapaverine hydrochloride; bone metabolism controlling, agents, such asvitamin D and active vitamin D3; sex hormones; hypotensives; anti-tumouragents; steroidal anti-inflammatory agents, such as hydrocortisone,prednisone, fluticasone, prednisolone, triamcinolone, triamcinoloneacetonide, dexamethasone, betamethasone, beclomethasone andbeclomethasone dipropionate; non-steroidal anti-inflammatory agents,such as acetaminophen, aspirin, aminopyrine, phenylbutazone, mefanicacid, ibuprofen, diclofenac sodium, indomethacin, colchicine andprobenecid; enzymatic anti-inflammatory agents, such as chymotrypsin andbromelain seratiopeptidase; anti-histaminic agents, such asdephenhydramine hydrochloride, chloropheniramine maleate and clemastine;anti-tussive-expectorants, such as codeine phosphate and isoproterenolhydrochloride; analgesics, such as opioids (like diamorphine, morphineand its polar metabolites, such as morphine-6-glucuronides andmorphine-3-sulphate); anti-emetics, such as metoclopramide, ondansetron,chlorpromazine; drugs for treatment of epilepsy, such as clonazepam;drugs for treatment of sleeping disorders, such as melatonin; drugs fortreatment of asthma, such as salbutamol.

Combinations of the abovementioned therapeutic agents may be employed.

The compositions according to the invention may be administered orally,nasally, vaginally, buccally, rectally, via the eye, or via thepulmonary route, in a variety of pharmaceutically acceptable dosingforms, which will be familiar to those skilled in the art. For example,compositions may be administered via the nasal route as a powder using anasal powder device, via the pulmonary route using a powder inhaler ormetered dose inhaler, via the vaginal route as a powder using a powderdevice, formulated into a vagina suppository or pessary or vaginaltablet or vaginal gel, via the buccal route formulated into a tablet ora buccal patch, via the rectal route formulated into suppositories; viathe eye in the form of a powder or a dry ointment, and via the oralroute in the form of a tablet, a capsule or a pellet (which compositionsmay administer agent via the stomach, the small intestine or the colon),all of which may be formulated in accordance with techniques which arewell known to those skilled in the art. The compositions may gel on themucosa at least to some extent and this may facilitate retention of thecomposition on the mucosa.

The preferred route of administration is nasal. Devices which may beused to deliver the compositions according to the invention nasallyinclude the Direct Haler®, the Bespak® powder device, the Monopoudre®(Valois) and the Insufflator® (Teijin).

Compositions according to the invention which may be administered orallymay be adapted to deliver therapeutic agent to the small intestine orthe colonic, especially the proximal colonic, region of thegastrointestinal tract.

Preferably, a means is provided to prevent release of therapeutic agentuntil the formulation reaches the small intestine or colon. Means whichmay be employed in order to prevent release until the small intestine isreached are well known to those skilled in the art (see for exampledosage forms coated with so-called enteric polymers that do not dissolvein the acidic conditions which exist in the stomach, but dissolve in themore alkaline conditions found in the small intestine of a mammal.Suitable enteric coating materials include modified cellulose polymersand acrylic polymers and in particular those sold under the trademarkEudragit®.) Means which may be employed in order to prevent releaseuntil the colon is reached are well known to those skilled in the art.Such materials include cellulose acetate trimellitate (CAT),hydroxypropylmethyl cellulose phthalate (HPMCP), polyvinyl acetatephthalate (PVAP), cellulose acetate phthalate (CAP) and shellac, asdescribed by Healy in his article “Enteric Coatings and DelayedRelease”, Chapter 7 in Drug Delivery to the Gastrointestinal Tract, eds.Hardy et al, Ellis Horwood, Chichester, 1989). Especially preferredmaterials are methylmethacrylates or copolymers of methacrylic acid andmethylmethacrylate. Such materials are available as Eudragit® entericpolymers (Rohm Pharma, Darmstadt, Germany). Such a coating may alsosuitably comprise a material which is redox-sensitive (e.g. azopolymerswhich may, for example, consist of a random copolymer of styrene andhydroxyetlyl methacrylate, cross-linked with divinylazobenzenesynthesised by free radical polymerisation, or disulphide polymers (seePCT/BE91/00006 and Van den Mooter, Int. J. Pharm. 87, 37(1992)). Seealso International Patent Application WO 97/05903.

It will be appreciated by those skilled in the art that the site ofdelivery may also be selectively controlled by varying the thickness ofcertain of the abovementioned polymer coatings.

It will be well understood by those skilled in the art that furtherexcipients may be employed in formulations comprising the compositionsaccording to the invention. For example, in solid dosing forms, furtherexcipients which may be employed include diluents such asmicrocrystalline cellulose (e.g. Avicel®, FMC), lactose, dicalciumphosphate and starch(es); disintegrants such as microcrystallinecellulose, starch(es) and cross-linked carboxymethylcellulose;lubricants such as magnesium stearatc and stearic acid; granulatingagents such as povidone; and release modifiers such as hydroxypropylmethylcellulose and hydroxypropyl cellulose. Suitable quantities of suchexcipients will depend upon the identity of the active ingredient(s) andthe particular dosing form which is used.

If desired, other materials may be included in the composition, forexample absorption enhancers. Suitable absorption enhancers includenon-ionic surfactants, cyclodextrins, bile salts and, preferably,phospholipids such as lysophosphatidylcholine, lysophosphatidylglyceroland generally those mentioned in WO 88/09163.

According to a further aspect of the invention, there is provided apharmaceutical formulation in a form suitable for administration to amucosal surface which comprises a composition according to the inventionin a pharmaceutically acceptable dosage form.

Compositions according to the invention have been found to have theadvantage that they provide improved transport of polar drugs acrossmucosal surfaces, such as the nasal cavity, have improved flowproperties when compared to prior art compositions, and avoid the needfor the use of chemical crosslinking agents.

According to a further aspect of the invention there is thus provided amethod for the improved transport of therapeutic agents across (or into)mucosal surfaces (which includes the presentation of vaccines to mucosalsurfaces) in mammals, and a method of treating a human or other mammal,which methods comprise administering a composition, as described above,preferably to a mucosal surface of that human or other mammal, forexample the vagina, buccal cavity, rectum, lungs, eye, colon, smallintestine, stomach or nasal cavity.

The amount of therapeutic agent which may be employed in thecompositions according to the invention will depend upon the agent whichis used. However, it will be clear to the skilled person that suitabledoses of therapeutic agents can be readily determined non-inventively.Suitable doses are in the range 1 μg to 1 g depending upon thetherapeutic agent(s) which is/are employed and the route ofadministration.

The invention is illustrated, but in no way limited, by the followingexamples with reference to the figures in which:

FIG. 1 shows the mean plasma glucose/time curves after administration tosheep of 2 IU/kg insulin in gelatin microspheres and in gelatin/chitosanmicrospheres containing either 9.6% or 19.28% G210 chitosan glutamate.

FIG. 2 shows the mean plasma insulin/time curves after administration tosheep of 2 IU/kg insulin in gelatin microspheres and in gelatin/chitosanmicrospheres containing either 9.6% or 19.28% G210 chitosan glutamate.

FIG. 3 shows the mean plasma calcium/time curves after administration tosheep of 20 IU/kg salmon calcitonin in gelatin microspheres and ingelatin/chitosan microspheres containing either 39.9% G110 or 19.9% G210chitosan glutamate.

FIG. 4 shows the mean plasma insulin/time curves after administration tosheep of 2 IU/kg insulin in 0.5% chitosan solution (G210) and ingelatin/chitosan microspheres containing 19.28% G210 chitosan glutamate.

FIG. 5 shows the mean plasma insulin/time curves after administration tosheep of 2 IU/kg insulin with chitosan powder (G210) and ingelatin/chitosan microspheres containing 19.28% G210 chitosan glutamate.

FIG. 6 shows the mean changes in plasma PTH concentration for aPTH/gelatin/chitosan microsphere formulation (PTH CHI/GER) as comparedto a formulation comprising PTH (alone) in saline (PTH sol) and PTH withchitosan glutamate (PTH CHI Sol).

FIG. 7 shows the effect on plasma glucose level of gelatin/chitosanmicrospheres comprising different amounts of insulin.

FIG. 8 shows the effect of repeated administration of gelatin/chitosanmicrospheres on plasma insulin level.

EXAMPLE 1

Preparation of Microspheres Containing 3.6% w/w Insulin, 86.7% w/wGelatin A and 9.6% w/w Chitosan Glutamate (Sea Cure G210)

193 mg chitosan glutamate was weighed into a 50 mL beaker and 15 mL ofwater was added and stirred until dissolution occurred. 1735 mg ofgelatin A (Sigma) was added to the chitosan solution and stirred at 40°C. until dissolution occurred. The pH of the solution was adjusted to 4by adding an appropriate amount of 1M HCl. 72 mg of human zinc insulin(1.8 mL of a 40 mg/mL insulin stock solution) was added to thegelatin/chitosan solution, which was transferred to a 20 mL volumetricflask and water added up to volume.

2 g of Span 80 was weighed into a metal beaker, 200 mL of sunflower oilwas added and the mixture warmed to 40° C. The 40° C.insulin/gelatin/chitosan solution was added and emulsified at 1000 rpmfor 5 minutes using a Heidolph stirrer fitted with a four blade stirrerarm maintaining the temperature at 40° C. The beaker was transferred toan ice bath and stirring continued at 1000 rpm until the temperature haddropped to below 10° C. The stirring speed was reduced to 500 rpm, 150mL of chilled acetone was added to the emulsion at 5 ml/min, and themixture was then centrifuged at 2500 rpm in centrifuge tubes for 10 min.The supernatant was discarded and the pellet resuspended in 50 mLacetone. The microspheres were recovered by vacuum filtration andwashing with further 50 mL of chilled acetone. The filter cake wasallowed to dry and the microspheres placed in 50 mL of acetone in ascrew capped bottle containing a magnetic stirrer and stirred overnight.The microspheres were vacuum filtered and dried in a desiccator.

EXAMPLE 2

Preparation of Microspheres Containing 3.6% w/w Insulin, 77.12% w/wGelatin A and 19.28% w/w Chitosan Glutamate (Sea Cure G210)

386 mg of chitosan glutamate was weighed into a 50 mL beaker, 15 mL ofwater was added and the resultant stirred until dissolution occurred.1542 mg of gelatin A was added to the chitosan solution, which was thenstirred at 40° C. until dissolution occurred. The pH of the solution wasadjusted to 4 by adding an appropriate amount of 1M HCl. 72 mg of humanzinc insulin (1.8 mL of a 40 mg/mL insulin stock solution) was added tothe gelatin/chitosan solution, which was then transferred to a 20 mLvolumetric flask, and water was added up to volume.

2 g of Span 80 was weighed into a metal beaker, 200 mL of sunflower oilwas added and the mixture warmed to 40° C. The 40° C.insulin/gelatin/chitosan solution was added and emulsified at 1000 rpmfor 5 minutes using a Heidolph stirrer fitted with a four blade stirrerarm maintaining the temperature at 40° C. The beaker was transferred toan ice bath and stirring continued at 1000 rpm until the temperature haddropped to below 10° C. The stirring speed was reduced to 500 rpm and150 mL of chilled acetone was added to the emulsion at 5 mL/min whichwas then centrifuged at 2500 rpm in centrifuge tubes for 10 min. Thesupernatant was discarded and the pellet resuspended in 50 mL acetone.The microspheres were recovered by vacuum filtration and washed withfurther 50 mL of chilled acetone. The filter cake was allowed to dry,the microspheres placed in 50 mL of acetone in a screw capped bottlecontaining a magnetic stirrer and stirred overnight. The microsphereswere vacuum filtered and dried in a desiccator.

EXAMPLE 3

Preparation of Microspheres Containing 0.2% w/w Salmon Calcitonin (SCT),59.9% w/w Gelatin A and 39.9% w/w Chitosan Glutamate (Sea Cure G110)

798 mg chitosan glutamate (G110) was weighed into a 50 mL beaker, 15 mLof water was added and the resultant stirred until dissolution occurred.1198 mg of gelatin A was added to the chitosan solution, which was thenstirred at 40° C. until dissolution occurred. The pH of the solution wasadjusted to 4 by adding an appropriate amount of 1M HCl. 20,000 IU ofSCT (0.91 mL of a 4 mg/mL SCT stock solution) was added to thegelatin/chitosan solution which was transferred to a 20 mL volumetricflask, and water was added up to volume.

2 g of Span 80 was weighed into a metal beaker, 200 mL of sunflower oilwas added and the mixture warmed to 40° C. The 40° C.SCT/gelatin/chitosan solution was added and emulsified at 1000 rpm for 5minutes using a Heidolph stirrer fitted with a four blade stirrer armmaintaining the temperature at 40° C. The beaker was transferred to anice bath and stirring continued at 1000 rpm until the temperature haddropped to below 10° C. The stirring speed was reduced to 500 rpm, 150mL of chilled acetone was added to the emulsion at 5 mL/min which wasthen centrifuged at 2500 rpm in centrifuge tubes for 10 min. Thesupernatant was discarded and the pellet resuspended in 50 mL acetone.The microspheres were recovered by vacuum filtration and washed with afurther 50 mL of chilled acetone. The filter cake was allowed to dry andthe microspheres placed in 50 mL of acetone in a screw capped bottlecontaining a magnetic stirrer and stirred overnight. The microsphereswere vacuum filtered and dried in a desiccator.

EXAMPLE 4

Preparation of Microspheres Containing 0.2% w/w SCT, 79.9% w/w Gelatin Aand 19.9% w/w Chitosan Glutamate (Sea Cure G210)

398 mg chitosan glutamate (G210) was weighed into a 50 mL beaker, 15 mLof water was added and the resultant mixture stirred until dissolutionoccurred. 1598 mg of gelatin A was added to the chitosan solution, whichwas stirred at 40° C. until dissolution occurred. The pH of the solutionwas adjusted to 4 by adding an appropriate amount of 1M HCl. 20,000 IUof SCT (0.91 mL of a 4 mg/mL SCT stock solution) was added to thegelatin/chitosan solution, which was then transferred to a 20 mLvolumetric flask and water was added up to volume.

2 g of Span 80 was weighed into a metal beaker, 200 mL of sunflower oilwas added and the mixture was warmed to 40° C. The 40° C.insulin/gelatin/chitosan solution was added and emulsified at 1000 rpmfor 5 minutes using a Heidolph stirrer fitted with a four blade stirrerarm, maintaining the temperature at 40° C. The beaker was transferred toan ice bath and stirring continued at 1000 rpm until the temperature haddropped to below 10° C. The stirring speed was reduced to 500 rpm, 150mL of chilled acetone was added to the emulsion at 5 mL/min which wasthen centrifuged at 2500 rpm in centrifuge tubes for 10 min. Thesupernatant was discarded and the pellet resuspended in 50 mL acetone.The microspheres were recovered by vacuum filtration and washed with afurther 50 mL of chilled acetone. The filter cake was allowed to dry andthe microspheres placed in 50 mL of acetone in a screw capped bottlecontaining a magnetic stirrer and stirred overnight. The microsphereswere vacuum filtered and dried in a desiccator.

EXAMPLE 5

The insulin-chitosan/gelatin microsphere formulations from Examples 1and 2 were administered nasally to sheep and the effect of theformulations was compared to the effect of administering insulin ingelatin A microspheres

The insulin—gelatin microspheres were prepared in the following way:1928 mg gelatin A was added to 14 mL of water in a 50 mL beaker andheated under stirring at 40° C. until the gelatin had dissolved. The pHof the gelatin solution was adjusted to 4 using 1M HCl and an equivalentof 72 mg of human zinc insulin (1.8 mL of 40 mg/mL insulin stocksolution) was added to the solution. The solution was transferred to a20 mL volumetric flask and made up to volume. 2 g of Span 80 was weighedinto a metal beaker, 200 mL of sunflower oil was added and the mixturewarmed to 40° C. The 40° C. insulin/gelatin solution was added andemulsified at 1000 rpm for 5 minutes using a Heidolph stirrer fittedwith a four blade stirrer arm, with the temperature maintained at 40° C.The beaker was transferred to an ice bath and stirring continued at 1000rpm until the temperature had dropped to below 10° C. The stirring speedwas reduced to 500 rpm and 150 mL of chilled acetone was added to theemulsion at 5 mL/min. The emulsion was centrifuged at 2500 rpm incentrifuge tubes for 10 min., the supernatant discarded and the pelletresuspended in 50 mL acetone. The microspheres were recovered by vacuumfiltration and washed with further 50 mL of chilled acetone. The filtercake was allowed to dry and the microspheres were placed in 50 mL ofacetone in a screw capped bottle containing a magnetic stirrer andstirred overnight. The microspheres were vacuum filtered and dried in adesiccator.

Each of the microsphere formulations were administered nasally to groupsof five sheep using blueline siliconised tubes at an insulin dose of 2IU/kg and a microsphere dose of 2.0 mg/kg. Blood samples were collectedat specified time points from the cannulated external jugular veins andplasma glucose and insulin concentrations measured. The mean changes inplasma glucose concentration with time for the three formulations areshown in FIG. 1. It can be seen that insulin given nasally incombination with gelatin microspheres did not result in any significantlowering of the plasma glucose levels (C_(min)=95.9%) whereas theformulations containing 9.6% chitosan and 19.28% chitosan gave glucoselowering effects of C_(min)=74.6% and C_(min)=53.8% of basal level,respectively. The corresponding plasma insulin levels for the threeformulations are shown in FIG. 2. It can be seen that C_(max) for boththe chitosan/gelatin microsphere formulations (131.6 mU/L and 439.7 mU/Lfor the 9.6/86.7% and 19.28/77.12% chitosan/gelatin microsphere,respectively) were significantly higher than the C_(max), seen for thegelatin microspheres (53.5 mU/L).

EXAMPLE 6

The calcitonin-chitosan/gelatin microsphere formulations described inExample 3 and 4 were administered nasally to sheep and the effect of theformulations compared to the effect of administering calcitonin ingelatin microspheres.

The calcitonin—gelatin microspheres were prepared in the following way:1996 mg gelatin A was added to 15 mL of water in a 50 mL beaker andheated under stirring at 40° C. until the gelatin had dissolved. The pHof the gelatin solution was adjusted to 4 using 1M HCl and an equivalentof 20,000 IU of salmon calcitonin (0.91 mL of 4 mg/mL SCT stocksolution) was added to the solution. The solution was transferred to a20 mL volumetric flask and made up to volume. 2 g of Span 80 was weighedinto a metal beaker, 200 mL of sunflower oil was added and the mixturewarmed to 40° C.

The 40° C. insulin/gelatin solution was added and emulsified at 1000 rpmfor 5 minutes using a Heidolph stirrer fitted with a four blade stirrerarm, with the temperature maintained at 40° C. The beaker wastransferred to an ice bath and stirring continued at 1000 rpm until thetemperature had dropped to below 10° C. The stirring speed was reducedto 500 rpm and 150 mL of chilled acetone was added to the emulsion at 5mL/min. The emulsion was centrifuged at 2500 rpm in centrifuge tubes for10 min., the supernatant was discarded and the pellet resuspended in 50mL acetone. The microspheres were recovered by vacuum filtration andwashed with further 50 mL of chilled acetone. The filter cake wasallowed to dry and the microspheres placed in 50 mL of acetone in ascrew capped bottle containing a magnetic stirrer and stirred overnight.The microspheres were vacuum filtered and dried in a desiccator.

Each of the microsphere formulations were administered nasally to groupsof five sheep using blueline siliconised tubes at an SCT dose of 20lU/kg and a microsphere dose of 2.004 mg/kg. Blood samples werecollected at specified time points from the cannulated external jugularveins and plasma calcium concentrations measured. The mean changes inplasma calcium concentration with time for the three formulations areshown in FIG. 3. It can be seen that SCT given nasally in combinationwith gelatin microspheres only resulted in a minimal lowering of theplasma calcium levels (C_(min)=91.1%) whereas the formulationscontaining 39.9% G110 chitosan and 19.9% G210 chitosan gave calciumlowering effects of C_(min)=73.6% and C_(min)=74.7% of basal level,respectively. There was no significant difference between the effectsobtained for the 39.9% G110 and 19.9% G210 chitosan levels in thegelatin microspheres.

EXAMPLE 7

The insulin-chitosan/gelatin microsphere formulation described inExample 2 was administered nasally to sheep and the effect of theformulation compared to the effect of administering insulin in a simplechitosan solution.

The microsphere formulation was administered nasally to a group of fivesheep using blueline siliconised tubes at an insulin dose of 2 IU/kg anda microsphere dose of 2.0 mg/kg. As a comparison, a solution of 200IU/mL insulin in 5 mg/mL G210 chitosan glutamate solution wasadministered nasally at 2 IU/kg to a group of four sheep. Blood sampleswere collected at specified time points from the cannulated externaljugular veins and plasma insulin concentrations measured. The meanchanges in plasma insulin concentration with time for the twoformulations are shown in FIG. 4. It can be seen that the plasma insulinlevel is significantly higher for the gelatin/chitosan microsphereformulation (C_(max)=450 mU/L) as compared to the chitosan solutionformulation (C_(max)=100 mU/L).

EXAMPLE 8

The insulin-chitosan/gelatin microsphere formulation described inExample 2 was administered nasally to sheep and the effect of theformulation compared to the effect of administering insulin with achitosan powder formulation.

The gelatin/chitosan microsphere formulation was administered nasally toa group of five sheep using blueline siliconised tubes at an insulindose of 2 IU/kg and a microsphere dose of 2.0 mg/kg. As a comparison, amixture of 640 IU insulin with 800 mg G210 chitosan glutamate wasadministered nasally at 2 IU/kg to a group of four sheep at 2 IU/kg.Blood samples were collected at specified time points from thecannulated external jugular veins and plasma insulin concentrationsmeasured. The mean changes in plasma insulin concentration with time forthe two formulations are shown in FIG. 5. It can be seen that the plasmainsulin level is significantly higher for the gelatin/chitosanmicrosphere formulation (C_(max)=450 mU/L) as compared to the chitosanpowder formulation (C_(max)250 mU/L). It should also be noted that theamount of chitosan administered in the two formulations is much higherfor the chitosan powder formulation than for the gelatin/chitosanmicrosphere formulation.

EXAMPLE 9

Preparation of Microspheres Containing 0.4% w/w PTH, 19.92%, w/wChitosan Glutamate (Sea Cure 210) and 79.68% w/w Gelatin A

9.28 mg of PTH was added to 20 mL of a solution containing 400 mgchitosan glutamate and 1.6 g of gelatin A and maintained at 50-60° C. 2g of Span 80 was weighed into a beaker and 200 mL of soya oil was added.The resultant was mixed and heated to 40° C. The PTH/chitosan/gelatinsolution was added and emulsified at 1000 rpm for 10 min. using aHeidolph stirrer fitted with a four blade stirrer arm, maintaining thetemperature at 40° C. The beaker was transferred to an ice bath andstirring continued at 100 rpm until the temperature had dropped to below10° C. The stirring speed was reduced to 500 rpm and 150 mL of chilledacetone was added to the emulsion at 5 mL/min, followed bycentrifugation at 3000 rpm for 10 min. The supernatant was discarded andthe pellet resuspended in acetone. The microspheres were recovered byvacuum filtration and washed with 50 mL of chilled acetone. The filtercake was allowed to dry, the microspheres placed in 50 mL of acetone ina screw capped bottle containing a magnetic stirrer and stirredovernight. The microspheres were vacuum filtered and dried in adissector.

Sheep Study

The PTH gelatin/chitosan microsphere formulation was administerednasally to a group of 6 sheep using blueline siliconised tubes at a PTHdose of 4 μg/kg. As a comparison, the same group of sheep was alsoadministered the same dose of PTH in saline and in saline containing0.5% chitosan glutamate. Blood samples were collected at specified timepoints from the cannulated external jugular veins and plasma PTHconcentrations measured. The mean changes in plasma PTH concentrationwith time for the three formulations are shown in FIG. 6. It can be seenthat the plasma PTH is significantly higher for the gelatin in chitosanmicrosphere formulation (C_(max)=2.5 ng/mL) as compared to the chitosansolution formulation (C_(max)=0.25 ng/mL) and the control PTH solution(C_(max)=0 ng/mL).

EXAMPLE 10

Determination of Hausner Ratio for Chitosan/Gelatin A Microspheres andFor Spray Dried Chitosan Microspheres (Sea Cure G210)

A known weight (see below) of chitosan/gelatin microspheres, prepared asin Example 2, was carefully poured into a 10 mL measuring cylinder andthe volume recorded (poured volume). The measuring cylinder was tapped(onto the bench) 50 times and the volume of the chitosan/gelatinmicrospheres again recorded (tapped volume). The measurement was carriedout in triplicate.

Poured Poured Tapped Tapped Hausner Weight Vol.(cm³) Den.(g/cm³)Vol.(cm³) Den. (g/cm³) Ratio 2.2481 7.3 0.3079 5.8 0.3876 1.26 2.36807.6 0.3116 6.0 0.3947 1.27 2.3220 7.5 0.3096 6.1 0.3807 1.23 HausnerRatio (chitosan/gelatin microspheres) = 1.25 (good flow porperties)

A known weight (see below) of spray dried chitosan microparticles (SeaCure G210; Pronova) was carefully poured into a 10 mL measuring cylinderand the volume recorded (poured volume). The measuring cylinder wastapped (onto the bench) 50 times and the volume of the chitosan againrecorded (tapped volume). The measurement was carried out in triplicate.

Poured Poured Tapped Tapped Hausner Weight Vol.(cm³) Den.(g/cm³)Vol.(cm³) Den. (g/cm³) Ratio 1.5609 9.0 0.1734 4.1 0.3807 2.20 1.47288.5 0.1733 3.8 0.3876 2.24 1.4004 8.0 0.1751 3.6 0.3890 2.22 HausnerRatio (chitosan) = 2.22 (very poor flow properties)

EXAMPLE 11

Determination of Angle of Repose For Chitosan/Gelatin A Microspheres andfor Spray Dried Chitosan Microspheres (Sea Cure G210)

The Angle of Repose (θ) was determined by pouring about 3 g ofchitosan/gelatin microspheres, prepared as in Example 2, through afunnel (held at a fixed height) onto a piece of graph paper until a conewas formed. The height (H) and the radius (R) of the cone weredetermined and the Angle calculated (tan θ=H/R). The measurement wascarried out in triplicate.

Mean Height=10 mm

Mean Radius=18 mm

Angle of Repose (chitosan/gelatin microspheres)=29° (good flowproperties)

The Angle of Repose (θ) was determined by pouring about 3 g of spraydried chitosan microparticles (Sea Cure G210; Pronova) through a funnel(held at a fixed height) onto a piece of graph paper until a cone wasformed. The height (H) and the radius (R) of the cone were determinedand the Angle calculated (tanθ=H/R). The measurement was carried out intriplicate.

Mean Height=25 mm

Mean Radius=24 mm

Angle of Repose (chitosan)=46° (very poor flow properties).

EXAMPLE 12

Determination of the Effect of Dose of Microspheres on the Absorption ofInsulin

Microspheres were prepared as in Example 2 with the final concentrationof insulin in the microspheres being 2.0%, 4.0%, 5.3%, 7.7% and 14.4%w/w.

The microspheres were administered nasally to groups of 4 sheep with afixed dose of 1 IU insulin/kg and 2.0, 1.0, 0.75, 0.5 and 0.25 mg/kg ofgelatin/chitosan microspheres as described in Example 8. The meanchanges in plasma glucose level expressed as AUC are given in FIG. 7. Itcan be seen that the effect of the gelatin/chitosan microsphercs on theAUC is no different whether 2.0 mg/kg or down to 0.25 mg/kg ofmicrospheres are administered with a constant dose of insulin.

EXAMPLE 13

Effect of Repeated Administration of Gelatin A/Chitosan Microspheres onPlasma Insulin Level

Gelatin/chitosan/insulin microspheres were prepared as in Example 2. Themicrospheres and a chitosan solution formulation, prepared as in Example8, were administered nasally to groups of 4 sheep once daily for 5consecutive days. A SC injection of insulin solution was given to thethird group of sheep for five consecutive days. Plasma insulin levelsexpressed as AUC are given in FIG. 8. It can be seen that the AUCobtained for each day is consistently higher for the gelatin/chitosanmicrosphere formulation as compared to the chitosan solutionformulation. It can also be seen that the AUCs obtained on the fiveconsecutive days are similar for the nasal formulation, thus showing aconsistent and reproducible effect, whereas a certain accumulativeeffect can be seen for the SC repeated injection.

We claim:
 1. A composition comprising a mixture of chitosan and type A,cationic, gelatin having an isoelectric point between pH 7 and 9; and atherapeutic agent.
 2. The composition of claim 1 wherein the compositionis in the form of microparticles.
 3. The composition of claim 2 whereinthe therapeutic agent is incorporated into the microparticles duringproduction of the microparticles, adsorbed to the surface of themicroparticles, or is present as an admixture with the microparticles.4. The composition of claim 2 wherein the microparticles aremicrospheres.
 5. The composition of claim 1 wherein the composition issuitable for delivery of the therapeutic agent across a mucosal membraneinto the systemic circulation.
 6. The composition of claim 1 wherein thechitosan has a molecular weight greater than 4000 Dalton.
 7. Thecomposition of claim 6 wherein the chitosan has a molecular weight inthe range 25,000 to 2,000,000 Dalton.
 8. The composition of claim 7wherein the chitosan has a molecular weight in the range 50,000 to30,000 Dalton.
 9. The composition of claim 1 wherein the chitosan is aderivative formed by bonding of acyl or alkyl groups with the hydroxylmoieties of the chitosan.
 10. The composition of claim 1 wherein thechitosan is in the form of a salt selected from the group consisting ofnitrates, phosphates, sulfates, hydrochloride, glutamates, lactate andacetate.
 11. The composition of claim 2 wherein the microparticles areproduced by a process selected from the group consisting of spraydrying, emulsification, solvent evaporation, and precipitation.
 12. Thecomposition of claim 1 wherein the chitosan has a degree ofdeacetylation of greater than 40%.
 13. The composition of claim 12wherein the degree of deacetylation is between 50 and 98%.
 14. Thecomposition of claim 13 wherein the degree of deacetylation is between70 and 90%.
 15. The composition of claim 2 wherein the microparticleshave a diameter of between 1 and 200 microns.
 16. The composition ofclaim 15 wherein the diameter is between 1 and 100 microns.
 17. Thecomposition of claim 1 comprising between 50 and 95% of type A gelatin.18. The composition of claim 17 comprising between 75 and 85% of type Agelatin.
 19. The composition of claim 1 wherein the therapeutic agent isa polar drug.
 20. The composition of claim 1 wherein the therapeuticagent is a polypeptide.
 21. The composition of claim 20 wherein thetherapeutic agent is selected from the group consisting of insulin,calcitonin, luteinising hormone releasing hormone, growth hormones, andgrowth hormone releasing factors.
 22. The composition of claim 1 whereinthe therapeutic agent is an analgesic agent or a drug for the treatmentof migraine.
 23. The composition of claim 1 wherein the therapeuticagent is an antigen intended for mucosal immunisation.
 24. Thecomposition of claim 1 wherein the therapeutic agent is a gene or geneconstruct (DNA) intended for the transfection of cells in the mucosalsurface.
 25. The composition of claim 1 further comprising an absorptionenhancing agent.
 26. The composition of claim 25 wherein the absorptionenhancing agent is a phospholipid.
 27. The composition of claim 1 in apharmaceutically acceptable dosage form suitable for administration to amucosal surface.
 28. A method for the improved transport of atherapeutic agent across a mucosal surface in a mammal, comprisingadministering a composition comprising (a) a mixture of chitosan andtype A cationic, gelatin, and (b) the therapeutic agent, to a mucosalsurface of the mammal.
 29. The composition of claim 27 for oraladministration wherein the mucosal surface is in the gastrointestinaltract.
 30. The method of claim 28 wherein the mucosal surface isselected from nasal mucosa, buccal cavity, vaginal mucosa, rectalmucosa, an eye, a lung, and gastrointestinal tract.
 31. A method oftreating a mammalian patient comprising administering to the patient acomposition comprising (a) a mixture of chitosan and type A, cationic,gelatin having an isoelectric point between pH 7 and 9, and (b)therapeutic agent.
 32. The method of claim 31 wherein the composition isdelivered to a mucosal surface.
 33. The method of claim 31 wherein thecomposition is adapted to deliver the therapeutic agent across a mucosalmembrane into the systemic circulation.
 34. A method for makingmicroparticles of type A gelatin and chitosan comprising spray drying amixture of chitosan and Type A, cationic gelatin having an isoelectricpoint between pH 7 and 9 to form the microparticles.
 35. A method formaking microparticles of type A gelatin and chitosan comprising mixing asolution of chitosan with type A, cationic gelatin having an isoelectricpoint between pH 7 and 9, which is warmed to melt the gelatin, to form amixture; emulsifying the mixture in a medium to form an emulsioncomprising microparticles of the mixture; and cooling the emulsion tosolidify the microparticles.
 36. The method of claim 35 wherein thechitosan is dissolved in water and mixed with the gelatin under heatingto 40° C.
 37. The method of claim 35 wherein the mixture is emulsifiedin the presence of an emulsifier, at a temperature above the meltingpoint of the gelatin, wherein the medium is an organic medium.
 38. Themethod of claim 35 wherein the microparticles are solidified bydecreasing the temperature of the emulsion below 10° C.
 39. The methodof claim 35 wherein the microparticles further comprise a therapeuticagent, the method further comprising incorporating the therapeutic agentinto the mixture before emulsification.
 40. The method of claim 34wherein the microparticles further comprise a therapeutic agent, themethod further comprising freeze drying or spray drying a suspension ofthe formed microparticles with the therapeutic agent, so that the agentis adsorbed onto the surface of the microparticles.
 41. The method ofclaim 34 wherein the microparticles further comprise a therapeuticagent, the method further comprising physically or mechanically mixingthe formed microparticles with the therapeutic agent, so that thetherapeutic agent is adsorbed onto the surface of the microparticles.42. The method of claim 35 wherein the microparticles further comprise atherapeutic agent, the method further comprising freeze drying or spraydrying a suspension of the solidified microparticles with thetherapeutic agent so that the therapeutic agent is adsorbed onto thesurface of the microparticles.
 43. The method of claim 35 wherein themicroparticles further comprise a therapeutic agent, the method furthercomprising isolating and drying the solidified microparticles and thenphysically or mechanically mixing the microparticles with thetherapeutic agent so that the therapeutic agent is adsorbed onto thesurface of the microparticles.